The determination of the stereochemistry and electronic structure of substrates and active site residues in reaction intermediates of enzymes stabilized by cryosolvent methods is proposed. Reaction intermediates stabilized ar subzero temperatures in organic-aqueous cosolvent mixtures will be prepared in solutions and in crystals for spectroscopic studies by ESR and ENDOR methods. The anisotropy of the ESR absorptions of paramagnetic centers and of the ENDOR of protons and other nuclides in the vicinity of the paramagnetic sites in single crystals will be analyzed to determine the precise geometrical relationships of substrates to catalytically essential active site residues. The methods are designed to determine local structure of enzymic active sites and of substrates in kinetically competent reaction intermediates to assess the structural and electronic basis of enzyme function on a detailed molecular level.